Multiline orthogonal scanning temporal focusing (mosTF) microscopy for scattering reduction in in vivo brain imaging

Temporal focusing two-photon microscopy has been utilized for high-resolution imaging of neuronal and synaptic structures across volumes spanning hundreds of microns in vivo. However, a limitation of temporal focusing is the rapid degradation of the signal-to-background ratio and resolution with increasing imaging depth. This degradation is due to scattered emission photons being widely distributed, resulting in a strong background. To overcome this challenge, we have developed multiline orthogonal scanning temporal focusing (mosTF) microscopy. mosTF captures a sequence of images at each scan location of the excitation line. A reconstruction algorithm then reassigns scattered photons back to their correct scan positions. We demonstrate the effectiveness of mosTF by acquiring neuronal images of mice in vivo. Our results show remarkable improvements in in vivo brain imaging with mosTF, while maintaining its speed advantage.